1. Field of the Invention
The present invention relates to regioselective and chemoselective hydrolysis of an .alpha.-ester group, of an amino acid diester using pig liver esterase enzyme (PLE). The amino acid diesters may be N-protected or unprotected and the ester groups may be the same or different.
2. Related Background Art
Throughout this application various publications are referenced by arabic numerals within parentheses (e.g., "Ref. 2"). Full citations for these references may be found at the end of the specification immediately preceding the claims. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
PLE has been used as a catalyst for the synthesis of chiral molecules via enantioselective ester hydrolysis (Ref. 1). PLE has also been used to catalyze the hydrolysis of cyclic meso diesters to prepare chiral half-esters (Ref. 2). Other uses of PLE have included the hydrolysis of ester groups of labile molecules in the synthesis of achiral molecules (Ref. 3).
Generally, acid-esters or half-esters may be prepared by any of the following methods: the esterification or transesterification of diacids with an alcohol in the presence of an acid catalyst (Refs. 5, 8a and 8b ); the hydrolysis of a cyclic anhydride with an alcohol (Refs. 6); or the hydrolysis of a diester with barium hydroxide (Ref. 7 and 9). It is often difficult to obtain a pure product (i.e. the monoester), using these methods due to the formation of a mixture of products formed during inefficient isolation procedures. The mixture of products may include the monoester, the diester, and the diacid. Additionally, these methods have the disadvantage of tending to give low yields of the monoester product.
The prior art reports several attempts to prepare amino acid monoesters by the selective hydrolysis of amino acid diesters Refs 4 and 11. Stein et al. demonstrated that the hydrolysis of aspartate dimethyl ester using PLE resulted in the hydrolysis of both ester groups, i.e. the .alpha.-ester group and the .beta.-ester group (Ref. 4). The selectivity of the .alpha.-ester hydrolysis to the .beta.-ester hydrolysis was found to be 98:2 for the formation of the corresponding aspartate monoesters In contrast, both the (R)-aspartate diethyl ester and (S)-aspartate diallyl ester are converted to their respective .beta.-monoesters with PLE with complete regioselectivity hydrolysis of the .alpha.-ester group. In addition, it was found that the preferential hydrolysis for the .alpha.-ester position is found to be partially reversed when the aspartate is N-protected as its formamide. The selectivity for the N-protected aspartate was found to be 55:45 for the .alpha.-ester hydrolysis to the .beta.-ester hydrolysis.
PLE has also been used by Adachi et al. in studying the regioselective hydrolysis of optically active unsymmetric diesters (Ref. 2h). In particular, Adachi et al. disclosed that the hydrolysis of dimethyl aspartate and dimethyl glutamate using crude PLE resulted in formation of a mixture of products due to hydrolysis of both ester groups. The hydrolysis of N-protected aspartates and N-protected glutamates using PLE also resulted in the formation of a mixture of products. Adachi et al. also reported that for the N-protected amino acid diesters hydrolysis was slower at the .alpha.-ester group than the .beta.- or .gamma.-ester groups. The faster hydrolysis of the N-protected aspartates and glutamates at the .beta.- and .gamma.-ester groups is believed to be due to the less crowded methyl ester group at these positions being preferably accommodated at the active site.
In addition to PLE other enzymes such as porcine pancreatic lipase (PPL) has been used for the regioselective hydrolysis of dialkyl amino acid esters (Ref. 10). Hydrolysis of N-protected dialkyl aspartates using PPL resulted in the regioselective hydrolysis of the .beta.-ester group and formation of the corresponding N-protected .alpha.-ester aspartate. However, in the case of unprotected dimethyl aspartate and glutamate esters, selective hydrolysis with PPL was not observed.
Accordingly, the development of an effective method for the regioselective hydrolysis of both u-protected and unprotected amino acid diesters remains a challenging problem.